EP3388523A1 - Enzymatisches verfahren zur herstellung von 2-hydroxy-4-methylmercaptobuttersäure (mha) - Google Patents

Enzymatisches verfahren zur herstellung von 2-hydroxy-4-methylmercaptobuttersäure (mha) Download PDF

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EP3388523A1
EP3388523A1 EP17166447.7A EP17166447A EP3388523A1 EP 3388523 A1 EP3388523 A1 EP 3388523A1 EP 17166447 A EP17166447 A EP 17166447A EP 3388523 A1 EP3388523 A1 EP 3388523A1
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dehydrogenase
mha
decarboxylase
hicdh
hydroxy
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Arne Skerra
Julia Martin
Harald Jakob
Daniel Fischer
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Technische Universitaet Muenchen
Evonik Operations GmbH
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Evonik Degussa GmbH
Technische Universitaet Muenchen
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Priority to EP18165674.5A priority patent/EP3388524B1/de
Priority to US15/947,824 priority patent/US10815508B2/en
Priority to JP2018076884A priority patent/JP2018174932A/ja
Priority to CN201810335751.7A priority patent/CN108728501A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01258—6-Hydroxyhexanoate dehydrogenase (1.1.1.258)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01001—Pyruvate decarboxylase (4.1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01043—Phenylpyruvate decarboxylase (4.1.1.43)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01072—Branched-chain-2-oxoacid decarboxylase (4.1.1.72)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
    • C12Y117/01—Oxidoreductases acting on CH or CH2 groups (1.17) with NAD+ or NADP+ as acceptor (1.17.1)

Definitions

  • the invention relates to an enzymatic method for producing 2-hydroxy-4-methylmercaptobutanoic acid from 3-methylthio-propanal (3-methylmercaptopropanal (MMP) or "methional").
  • 2-Hydroxy-4-methylmercaptobutanoic acid is used as a feed additive in a similar way to methionine and, owing to the structural similarity, is therefore known as methionine hydroxy analog (MHA).
  • MHA methionine hydroxy analog
  • methional which, in turn, is obtainable by addition of methyl mercaptan (methanethiol) to acrolein, followed by reaction with hydrogen cyanide and subsequent hydrolysis of the 4-methylmercapto-2-hydroxybutanenitrile formed.
  • hydrogen cyanide is a disadvantage of this process. Owing to the high toxicity of hydrogen cyanide, outlay on safety must be high for the reaction.
  • Burrington et al. (US 4,782,173 ) propose a three-step catalytic conversion of methional to MHA using carbon monoxide as C1 building block.
  • Lehmann et al. (WO 02/16671 A1 ) disclose a process for the production of MHA by electrochemical carboxylation of methional in an undivided electrolytic cell containing a sacrificial anode, an aprotic solvent and a supporting electrolyte using carbon dioxide as C1 building block.
  • a fermentative method for producing MHA by culturing microorganisms under nitrogen limitation is proposed by Figge et al. (WO 2012/090022 A1 ).
  • the microorganisms produce methionine, which under nitrogen limitation is used as a nitrogen source to yield its deamination product 4-methylthio-2-oxobutanoic acid (MTOB) and MHA by reduction of MTOB ( Ince and Knowles (1986) Arch. Microbiol. 146,151-158 ).
  • MTOB 4-methylthio-2-oxobutanoic acid
  • Miyazaki et al. (Chem. Commun. (2001) 1800-1801 ) propose the reverse reaction of pyruvate decarboxylase using carbon dioxide as C1 building block for the synthesis of pyruvic acid (2-oxopropanoic acid) from ethanal (acetaldehyde).
  • the reaction requires a large excess of bicarbonate as carbon dioxide source in order to drive the equilibrium into the opposite direction of decarboxylation.
  • a multienzyme catalytic system including a cofactor regeneration cycle that uses a carbonate-bicarbonate buffer and ethanol to produce L-lactate via acetaldehyde and pyruvic acid was proposed by Tong et al. (Biotechnol. Bioeng. (2011) 108, 465-469 ).
  • the object of the present invention is to provide a novel process for the production of MHA, in which, on the one hand, methional is used as a starting component and, on the other hand, instead of HCN carbon dioxide is used as C1 building block yielding D- or L-MHA.
  • the object is achieved by a method for producing D- or L-2-hydroxy-4-methylmercaptobutanoic acid (MHA), comprising a step of reacting a mixture comprising 3-(methylthio)-propanal (methional), carbon dioxide, a decarboxylase, its corresponding cofactor, an alcohol dehydrogenase and NADH or NADPH to form D- or L-2-hydroxy-4-methylmercaptobutanoic acid (MHA) or a salt thereof.
  • MHA D- or L-2-hydroxy-4-methylmercaptobutanoic acid
  • the decarboxylase in the mixture catalyzes its reverse reaction, i.e. the carboxylation of methional with carbon dioxide (CO 2 ), which leads to the intermediate product 4-methylthio-2-oxobutanoic acid (MTOB), i.e. an ⁇ -keto acid (2-oxo acid).
  • MTOB 4-methylthio-2-oxobutanoic acid
  • the ⁇ -carbonyl group of MTOB is reduced to the hydroxyl group in a stereospecific and NADH- (or, alternatively, NADPH-) dependent reaction catalyzed by an alcohol dehydrogenase to yield MHA.
  • Figure 1 shows a scheme for this two-step biocatalytic synthesis of MHA from methional.
  • the corresponding cofactor of the decarboxylase preferably comprises thiamine pyrophosphate (ThDP).
  • Decarboxylases that are suitable for the carboxylation of methional are for example pyruvate decarboxylase Pdc1, which originates from Saccharomyces cerevisiae, phenylpyruvate decarboxylase Aro10, which originates from Saccharomyces cerevisiae, and branched chain decarboxylase KdcA, which originates from Lactococcus lactis, as well as mutants and variants of these decarboxylase having decarboxylase activity.
  • Pdc1 pyruvate decarboxylase
  • Aro10 which originates from Saccharomyces cerevisiae
  • KdcA branched chain decarboxylase
  • the method according to the present invention is suitable for producing D-2-hydroxy-4-methylmercapto-butanoic acid (D-MHA) as well as L-2-hydroxy-4-methylmercapto-butanoic acid (L-MHA).
  • D-MHA D-2-hydroxy-4-methylmercapto-butanoic acid
  • L-MHA L-2-hydroxy-4-methylmercapto-butanoic acid
  • a suitable alcohol dehydrogenase is for example a D-hydroxyisocaproate dehydrogenase, preferably D-HicDH from Lactobacillus casei or its mutants and variants having alcohol dehydrogenase activity
  • L-hydroxyisocaproate dehydrogenase preferably L-HicDH from Lactobacillus confusus or its mutants and variants having alcohol dehydrogenase activity may for example be chosen as an alcohol dehydrogenase.
  • the carbon dioxide is preferably applied to the reaction mixture at a pressure from 10 to 7400 kPa (from 0,1 to 74 bar), preferably from 100 to 1000 kPa (1 to 10 bar), more preferably from 200 to 800 kPa (2 to 8 bar).
  • the mixture may further comprise formic acid or a salt thereof and a formate dehydrogenase.
  • regeneration of the NADH consumed in the method according to the present invention can be achieved under consumption of formate and formation of carbon dioxide ( Figure 1 ).
  • the carbon dioxide may serve as substrate for the reverse decarboxylation reaction described above.
  • Suitable formate dehydrogenases are for example formate dehydrogenase from Pseudomonas sp., e.g. formate dehydrogenase PseFDH from Pseudomonas sp. 101, and formate dehydrogenase from Candida sp., e.g. formate dehydrogenase from Candida boidinii optionally carrying the amino acid substitutions C23A and F285S, as well as mutants and variants of these formate dehydrogenases having formate dehydrogenase activity.
  • Table 1 Enzymes suitable for the synthesis of MHA from Methional Enzyme class and example Organism Modification(s) Quaternary structure SEQ ID No. Reaction Decarboxylase EC 4.1.1 Pyruvate decarboxylase: Pdc1 (P06169; Killenberg-Jabs et al. (1997) Biochemistry 36, 1900-1905 ) Saccharomyces cerevisiae C-terminal His 6 -tag tetramer 1 Carboxylation of Methional to MTOB Phenylpyruvate decarboxylase: Aro10 (Q06408; Kneen et al. (2011) FEBS J.
  • Lactobacillus casei N-terminal His 6 -tag dimer 7 Reduction of MTOB to D-MHA L-Hydroxyisocaproate dehydrogenase: L-HicDH (P14295; Scbou et al. (1984) Appl. Microbiol. Biotechnol. 19, 167-176 ) Lactobacillus confusus C-terminal Hiss-tag tetramer 9 Reduction of MTOB to L-MHA Formate dehydrogenase EC 1.2.1.2 Formate dehydrogenase: PseFDH (P33160; Egorov et al. (1979) Eur. J. Biochem.
  • N-terminal His 6 -tag dimer 11 NADH-regeneration from NAD + by the oxidation of formate to CO 2 Formate dehydrogenase optionally carrying the amino acid substitutions C23A and F285S: CboFDH(C23A/F285S) (013437 for the wild type enzyme; Scbou et al. (1976) Eur. J. Biochem. 62, 151-160 ; Slusarczyk et al. (2000) Eur. J. Biochem, 267, 1280-1287 ; Felber (2001) Doctoral Thesis, Heinrich-Heine University Düsseldorf ) Candida boidinii C23A/F285S; N-terminal His 6 -tag dimer 13
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • decarboxylases were purified using a two-step strategy comprising an immobilized metal ion affinity chromatography (IMAC) followed by a size exclusion chromatography (SEC). Therefore, the cells were resuspended in 3 ml 300 mM NaCl, 1 mM MgSO 4 , 0.1 mM thiamine pyrophosphate (ThDP), 20 mM PIPES/NaOH pH 7.0 per 1 g wet weight and then disrupted mechanically using a French pressure cell (SLM Aminco, Rochester, NY).
  • IMAC immobilized metal ion affinity chromatography
  • SEC size exclusion chromatography
  • the homogenate was centrifuged (30 min, 18000 rpm, fixed angle rotor, 4 °C), and the complete supernatant was applied to a 5 ml bed volume HisTrap HP column (GE Healthcare, Munich, Germany) charged with Ni(II) ions using 300 mM NaCl, 1 mM MgSO 4 , 0.1 mM ThDP, 20 mM PIPES/NaOH pH 7.0 as running buffer.
  • the bound decarboxylase was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer.
  • Main fractions containing the decarboxylase were identified by Commassie-stained SDS-PAGE and concentrated to a final volume of 2-2.5 ml using a centrifugal filter unit with a nominal molecular weight limit (NMWL) of 30 kDa (Merck, Darmstadt, Germany).
  • NMWL nominal molecular weight limit
  • the concentrated sample was further purified via SEC using a 120 ml bed volume HiLoad Superdex 200 16/60 column (GE Healthcare) in the presence of 500 mM NaCl, 1 mM MgSO 4 , 0.5 mM ThDP, 20 mM PIPES/NaOH pH 7.0.
  • D-HicDH D-hydroxyisocaproate dehydrogenase from Lactobacillus casei (SEQ ID NO: 7; P17584; Hummel et al. (1985) Appl. Microbiol. Biotechnol. 21, 7-15 ) was synthesized with optimal codon usage for expression in E. coli (Geneart) and cloned on the expression vector pASK-IBA35(+) (IBA, Göttingen, Germany) using the restriction enzymes Kasl and Hin dIII.
  • D-HicDH D-hydroxyisocaproate dehydrogenase
  • the synthesized gene (SEQ ID NO: 9) was cloned on the expression vector pASK75(T7RBS)his using the restriction enzymes Nde I and Eco 47III.
  • the temperature was reduced to 22 °C during 45-60 min, while for the production of the L-HicDH the temperature was kept at 30 °C.
  • aTc anhydrotetracycline
  • the bacteria were harvested by centrifugation (10 min, 6000 rpm, fixed angle rotor, 4 °C) and frozen at -20 °C.
  • the cells containing the D-HicDH were resuspended in 3 ml 150 mM NaCl, 50 mM PIPES pH 7.0 per 1 g wet weight while the cells containing L-HicDH were resuspended in 3 ml 300 mM NaCl, 50 mM KP i pH 7 per 1 g wet weight. Then the bacteria were disrupted mechanically in a French pressure cell.
  • the homogenate was centrifuged (30 min, 18000 rpm, fixed angle rotor, 4 °C) and the entire supernatant was applied to a 5 ml bed volume HisTrap HP column charged with Ni(II) ions using 150 mM NaCl, 50 mM PIPES pH 7.0 for the D-HicDH and 300 mM NaCl, 50 mM KP i pH 7 for the L-HicDH, respectively, as running buffer.
  • the bound dehydrogenase was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer.
  • Main fractions containing the dehydrogenase were identified by Commassie-stained SDS-PAGE and concentrated to a final volume of 4-5 ml using a centrifugal filter unit with a NMWL of 30 kDa.
  • the concentrated sample was purified by SEC using a 320 ml bed volume HiLoad Superdex 200 26/60 column in the presence of 150 mM NaCl, 50 mM PIPES pH 7.0 and 300 mM NaCl, 20 mM KP i pH 6.5 for D-HicDH and L-HicDH, respectively.
  • the gene for the formate dehydrogenase from Pseudomonas sp. 101 was synthesized with optimal codon usage for expression in E. coli (Geneart) and cloned on the expression vector pASK-IBA35(+) using the restriction enzymes Kasl and Hin dIII .
  • the gene for the formate dehydrogenase from Candida boidinii was synthesized with optimal codon usage for expression in E. coli (Geneart) and cloned on the expression vector pASK-IBA35(+) using the restriction enzymes Kasl and Hin dIII .
  • the gene for the formate dehydrogenase from Candida boidinii CboFDH; 013437; Scrissa et al. (1976) Eur. J. Biochem.
  • the bacterial paste was resuspended in 3 ml per 1 g wet cell mass in 300 mM NaCl, 50 mM KPi pH 7.5 and disrupted mechanically using a French pressure cell. After centrifugation (30 min, 18000 rpm, fixed angle rotor, 4 °C), the entire supernatant was applied to a 5 ml bed volume HisTrap HP column charged with Ni(II) ions using 300 mM NaCl, 50 mM KPi pH 7.5 as running buffer. The bound FDH was eluted by a linear concentration gradient of 0 to 500 mM imidazole/HCl in running buffer.
  • the eluted protein was concentrated using a centrifugal filter unit with a NMWL of 30 kDa.
  • the formate dehydrogenases PseFDH and CboFDH(C23A/F285S) were obtained in yields of 19 mg/l and 45 mg/l, respectively. High purity of > 95 % was confirmed by Commassie-stained SDS-PAGE analysis.
  • Example 4 Synthesis of the D-methionine-hydroxy-analog (D-MHA) from 3-(methylthio)propanal (methional) by a two-step biocatalytic reaction involving a decarboxylase and an alcohol dehydrogenase.
  • D-MHA D-methionine-hydroxy-analog
  • the carboxylation reaction under catalysis of the decarboxylase KdcA was started by the addition of the substrate methional and application of 200 kPa (2 bar) CO 2 .
  • the initial pH of the mixture was 8, which shifted to ca. 6.5 upon application of CO 2 (as measured with a fixed-color-pH indicator stick (Carl Roth, Düsseldorf, Germany) in a sample).
  • the mixture was recovered from the reactor and centrifuged for 5 min at 13400 rpm in a bench top centrifuge to remove precipitated proteins.
  • product formation was analyzed by HPLC using a C18 column (Gemini C18, 4.6x15 mm, 3 ⁇ m, 110 A; Phenomenex, Aillesburg, Germany) with isocratic elution in 4 % (v/v) aqueous acetonitrile supplemented with 1 % (v/v) phosphoric acid.
  • Example 5 Synthesis of D-MHA from Methional by a two-step biocatalytic reaction involving a decarboxylase and an alcohol dehydrogenase in the presence of a biocatalytic NADH regeneration system.
  • D-MHA from methional catalyzed by a decarboxylase (e.g. KdcA) and an alcohol dehydrogenase (e.g. D-HicDH)
  • a decarboxylase e.g. KdcA
  • an alcohol dehydrogenase e.g. D-HicDH
  • the cosubstrate NADH is consumed by the dehydrogenase for reduction of the ⁇ -carbonyl group of MTOB.
  • a formate dehydrogenase e.g. CboFDH(C23A/F285S)
  • CboFDH(C23A/F285S) can be employed.
  • This enzyme oxidizes formate with NAD+ as cosubstrate to yield CO2, which may also serve as substrate for the carboxylation reaction of methional, as well as NADH ( Scrissa et al. (1976) Eur. J. Biochem. 62, 151-160 ; Wichmann et al. (1981) Biotechnol. Bioeng. 23, 2789-2802 ).
  • This three-enzyme coupled reaction was performed under a limiting concentration of NADH (80 ⁇ M), which was added to a reaction mixture in a 10 ml pressure reactor (Tinyclave steel) containing the following reagents in a final volume of 1 ml: Reagent/enzyme Final concentration NaHCO3 200 mM ThDP 0.5 mM MgCl2 1 mM KdcA 10 ⁇ M D-HicDH 0.5 ⁇ M CboFDH(C23A/F285S) 10 ⁇ M NADH 80 ⁇ M NaHCO3 25 mM Methional 4 mM
  • Example 4 As in Example 4 the reaction was started by the addition of methional and application of 800 kPa (8 bar) CO 2 . The initial pH of the mixture was 8 and shifted to ca. 6.5 upon application of CO 2 (as measured with a fixed-color- pH indicator stick in a sample). After 1.5 h incubation, the mixture was recovered from the reactor and analyzed using HPLC as described in Example 4. The resulting chromatogram showed a significantly increased D-MHA peak ( ⁇ 114 ⁇ M) compared to the control reaction ( ⁇ 38 ⁇ M) in which the substrate formate of the formate dehydrogenase was omitted ( Fig. 2 D) .
  • a formate dehydrogenase together with its substrate to the D-MHA-forming reaction involving a decarboxylase (e.g, KdcA) and a NADH dependent alcohol dehydrogenase (e.g. D-HicDH), can compensate for limiting NADH concentrations and regenerate this cosubstrate.
  • a decarboxylase e.g, KdcA
  • a NADH dependent alcohol dehydrogenase e.g. D-HicDH
  • Example 6 Synthesis of the L-methionine-hydroxy-analog (L-MHA) from Methional by a two-step biocatalytic reaction involving a decarboxylase and an alcohol dehydrogenase.
  • the purified decarboxylase e.g. KdcA
  • the alcohol dehydrogenase e.g. L-HicDH
  • Reagent/enzyme Final concentration NaHCO3 200 mM ThDP 0.5 mM MgCl2 1 mM KdcA 20 ⁇ M L-HicDH 500 nM NADH 4 mM Methional 4 mM
  • the reaction was started by the addition of methional and application of 800 kPa (8 bar) CO 2 as described herein above in Examples 4 and 5. Upon the application of CO 2 the initial pH of 8 was shifted to 6.5 as measured with a fixed-color-pH indicator stick in a sample.

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EP17166447.7A 2017-04-13 2017-04-13 Enzymatisches verfahren zur herstellung von 2-hydroxy-4-methylmercaptobuttersäure (mha) Withdrawn EP3388523A1 (de)

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EP17166447.7A EP3388523A1 (de) 2017-04-13 2017-04-13 Enzymatisches verfahren zur herstellung von 2-hydroxy-4-methylmercaptobuttersäure (mha)
EP18165674.5A EP3388524B1 (de) 2017-04-13 2018-04-04 Enzymatisches verfahren zur herstellung von 2-hydroxy-4-methylmercaptobuttersäure (mha)
US15/947,824 US10815508B2 (en) 2017-04-13 2018-04-08 Enzymatic method for producing 2-hydroxy-4-methylmercaptobutanoic acid (MHA)
JP2018076884A JP2018174932A (ja) 2017-04-13 2018-04-12 ２−ヒドロキシ−４−メチルメルカプト酪酸（ｍｈａ）の酵素的製造方法
CN201810335751.7A CN108728501A (zh) 2017-04-13 2018-04-13 生产2-羟基-4-甲硫基丁酸(mha)的酶促方法

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EP3330380A1 (de) * 2016-12-05 2018-06-06 Evonik Degussa GmbH Verfahren zur herstellung von l-methionin aus methional
CN109593702B (zh) * 2019-01-17 2020-10-09 江南大学 一种基因工程菌株实现全细胞转化合成l-苯乳酸的方法
EP4121542A4 (de) * 2020-03-18 2024-05-15 Lanzatech, Inc. Fermentative herstellung von 2-phenylethanol aus gasförmigen substraten
CN113845454A (zh) * 2020-06-28 2021-12-28 张科春 一种酮基蛋氨酸及其衍生物的制备方法及应用
CN117603924B (zh) * 2023-04-25 2024-05-17 杭州力文所生物科技有限公司 蛋白可溶性表达提高的甲酸脱氢酶突变体及其应用

Citations (5)

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US4782173A (en) 1983-08-03 1988-11-01 The Standard Oil Company Synthesis of methionine hydroxy analog or derivative, and esters thereof; synthesis of 1-acyloxy-4-hydrocarbylthiopropene, and products
WO2002016671A1 (en) 2000-08-18 2002-02-28 Degussa Ag Process for the production of 2-hydroxy-4-methylmercaptobutyric acid
US20030157664A1 (en) 2001-09-21 2003-08-21 Degussa Ag Novel mutants of the formate dehydrogenase from Candida boidinii
WO2012090022A1 (en) 2010-12-30 2012-07-05 Metabolic Explorer Fermentative production of methionine hydroxy analog (mha)
WO2017005910A1 (en) * 2015-07-09 2017-01-12 Metabolic Explorer Methionine hydroxy analog (mha) pathways for production by fermentation

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